Novel Antioxidant Therapy with the Immediate Precursor to Glutathione, γ-Glutamylcysteine (GGC), Ameliorates LPS-Induced Cellular Stress in In Vitro 3D-Differentiated Airway Model from Primary Cystic Fibrosis Human Bronchial Cells

Chris K Hewson; Alexander Capraro; Sharon L Wong; Elvis Pandzic; Ling Zhong; Bentotage S M Fernando; Nikhil T Awatade; Gene Hart-Smith; Renee M Whan; Shane R Thomas; Adam Jaffe; Wallace J Bridge; Shafagh A Waters

doi:10.3390/antiox9121204

Novel Antioxidant Therapy with the Immediate Precursor to Glutathione, γ-Glutamylcysteine (GGC), Ameliorates LPS-Induced Cellular Stress in In Vitro 3D-Differentiated Airway Model from Primary Cystic Fibrosis Human Bronchial Cells

Antioxidants (Basel). 2020 Nov 30;9(12):1204. doi: 10.3390/antiox9121204.

Authors

Chris K Hewson^{1

2

3}, Alexander Capraro^{1

2}, Sharon L Wong^{1

2}, Elvis Pandzic⁴, Ling Zhong⁵, Bentotage S M Fernando⁶, Nikhil T Awatade^{1

2}, Gene Hart-Smith^{3

7}, Renee M Whan⁴, Shane R Thomas⁶, Adam Jaffe^{1

2

8}, Wallace J Bridge³, Shafagh A Waters^{1

2

8}

Affiliations

¹ School of Women's and Children's Health, Faculty of Medicine, University of New South Wales, Sydney, NSW 2052, Australia.
² Molecular and Integrative Cystic Fibrosis (miCF) Research Centre, University of New South Wales and Sydney Children's Hospital, Sydney, NSW 2052, Australia.
³ School of Biotechnology and Biomolecular Sciences, Faculty of Science, University of New South Wales, Sydney, NSW 2052, Australia.
⁴ Biomedical Imaging Facility, University of New South Wales, Sydney, NSW 2052, Australia.
⁵ Bioanalytical Mass Spectrometry Facility, University of New South Wales, Sydney, NSW 2052, Australia.
⁶ School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, NSW 2052, Australia.
⁷ Department of Molecular Sciences, Faculty of Science and Engineering, Macquarie University, Sydney, NSW 2109, Australia.
⁸ Department of Respiratory Medicine, Sydney Children's Hospital, Sydney, NSW 2031, Australia.

Abstract

Systemic glutathione deficiency, inflammation, and oxidative stress are hallmarks of cystic fibrosis (CF), an inherited disease that causes persistent lung infections and severe damage to the respiratory system and many of the body organs. Improvements to current antioxidant therapeutic strategies are needed. The dietary supplement, γ-glutamylcysteine (GGC), which is the immediate precursor to glutathione, rapidly boosts cellular glutathione levels following a single dose in healthy individuals. Efficacy of GGC against oxidative stress induced by Pseudomonas aeruginosa, which is a common and chronic pathogen infecting lungs of CF patients, remains unassessed. Primary mucocilliary differentiated airway (bronchial and/or nasal) epithelial cells were created from four individuals with CF. Airway oxidative stress and inflammation was induced by P. aeruginosa lipopolysaccharide (LPS). Parameters including global proteomics alterations, cell redox state (glutathione, oxidative stress), pro-inflammatory mediators (IL-8, IDO-1), and cellular health (membrane integrity, stress granule formation, cell metabolic viability) were assayed under six experimental conditions: (1) Mock, (2) LPS-challenged (3) therapeutic, (4) prophylactic (5) therapeutic and prophylactic and (6) GGC alone. Proteomic analysis identified perturbation of several pathways related to cellular respiration and stress responses upon LPS challenge. Most of these were resolved when cells were treated with GGC. While GGC did not resolve LPS-induced IL-8 and IDO-1 activity, it effectively attenuated LPS-induced oxidative stress and stress granule formation, while significantly increasing total intracellular glutathione levels, metabolic viability and improving epithelial cell barrier integrity. Both therapeutic and prophylactic treatments were successful. Together, these findings indicate that GGC has therapeutic potential for treatment and prevention of oxidative stress-related damage to airways in cystic fibrosis.

Keywords: LPS; Pseudomonas aeruginosa; antioxidant; cystic fibrosis; glutathione; oxidative stress; redox.